Process for the preparation of immobilized and activity-stabilized complexes of LHRH antagonists

ABSTRACT

In this invention, a release-delaying system is to be developed for LHRH antagonists, in particular for cetrorelix, which allows the active compound to be released in a controlled manner over several weeks by complexation with suitable biophilic carriers. The acidic polyamino acids polyglutamic acid and polyaspartic acid were selected for complexation with cetrorelix. The cetrorelix polyamino acid complexes are prepared from aqueous solutions by combination of the solutions and precipitation of the complexes, which are subsequently centrifuged off and dried over P 2  O 5  in vacuo. If complexes having a defined composition are to be obtained, lyophilization proves to be a suitable method. The cetrorelix-carboxylic acid complexes were also prepared from the aqueous solutions. In the random liberation system, the acidic polyamino acids poly-Glu and poly-Asp showed good release-delaying properties as a function of the hydrophobicity and the molecular mass of the polyamino acid. In animal experiments, it was possible to confirm the activity of the cetrorelix-polyamino acid complexes as a depot system in principle. It is thus possible by complexation of cetrorelix with polyamino acids to achieve testosterone suppression in male rats over 600 hours. The release of active compound here can be controlled by the nature and the molecular mass of the polymers.

This is a division of Application No. 09/048,244, filed Mar. 26, 1998,now pending.

BACKGROUND OF THE INVENTION

The invention relates to activity-stabilized and release-delayingcomplexes of LHRH antagonists such as antide, antarelix, azaline,A-75998, ganirelix, Nal--Glu antagonist with polyamino acids, inparticular polyglutamic acid and polyaspartic acid, and processes forthe preparation thereof and pharmaceuticals comprising these.

The peptide hormone-polyamino acid complexes prepared can be used inmedicine, for example for the therapy of hormone-sensitive tumours, suchas, for example, breast and prostate carcinoma, benign prostatehypertrophy and in gynaecology for the treatment of endometriosis,hysteroscopy and for the treatment of fertility disorders.

BACKGROUND INFORMATION

In the Patent Specifications DD 257197, DD 269785 and DD 299265, forinsulin and for other biologically active proteohormones, processes forthe preparation of immobilized peptide preparations stabilized in theirbiological activities and modified in their pharmacological propertiesare described, whose most important feature is the complexation of therespective peptide with polyamino acids.

In the patents mentioned, preparation procedures are described in whichthe complexes are formed under the action of formic acid and organicsolvents such as chloroform and drastic preparation conditions. There isthe danger for the peptide hormone in these processes of partialinactivation and decreased stability.

In the literature, in 1981 for the first time, poorly soluble salts orcomplexes of LHRH analogues were described in EP 0042753 and EP 0049628.The preparation of these complexes was carried out with a view to thedevelopment of pharmaceutical products for different medicinalapplications.

In 1989, ORSOLINI in DE Patent 38 22 459 describes the preparation ofwater-insoluble polypeptides by complexation of LHRH analogues withembonic acid, tannin and stearic acid. The poorly soluble complexesobtained are in this case additionally embedded in a polymeric matrix of(lactic acid-glycolic acid) copolymer.

Further processes for the preparation of cetrorelix complexes embeddedin a (lactic acid-glycolic acid) copolymer were described in 1993 byORSOLINI and HEIMGARTNER in DE Patents 42 23 282 and 42 23 284. In thispatent, poorly soluble cetrorelix complexes with embonic acid, tannin,stearic acid and palmitic acid [lacuna] mentioned.

SUMMARY OF THE INVENTION

The aim of the invention is to prepare depot preparations havingimproved and controllable release-delaying properties and increasedstability against premature proteolytic degradation of LHRH antagonistsfor therapy in the areas known for this such as hormone-sensitivetumours, such as, for example, breast and prostate carcinoma, benignprostate hypertrophy, endometriosis, hysteroscopy and for the treatmentof fertility disorders and to indicate an easily controllable andenvironmentally friendly process for the production of thesepreparations.

The object of the invention is to prepare novel depot preparationshaving improved and controllable release-delaying properties of LHRHantagonists such as antide, antarelix, azaline, A-75998, ganirelix,Nal--Glu antagonist, but preferably cetrorelix, with biodegradablepolymers, and a process for their preparation.

According to the invention, the object is achieved by preparingimmobilized and activity-stabilized, parenterally administerable peptidehormone preparations from complexes of LHRH antagonists with polyaminoacids, in particular polyglutamic acid and polyaspartic acid byprecipitating the polyamino acid-peptide hormone complex from aqueoussolutions avoiding organic solvents. Advantageously, the polyaminoacid-peptide hormone complexes can furthermore be prepared with acontrollable hormone content by lyophilization of the aqueous solutions.By means of the nature and the molecular mass of the polyamino acids, byincorporation of hydrophobic amino acids into the polymer structure orby partial esterification, the release rate of the active compound canbe controlled (FIGS. 2 and 3).

The complexes according to the invention are used in medicine for thetherapy of hormone-sensitive tumours, in particular for the treatment ofbreast and prostate carcinomas, of benign prostate hypertrophy and ingynaecology for the induction of ovulation, in vitro fertilization andendometriosis and in connection with hysteroscopy.

The term "complex" in the context of this invention comprises theassembly of two or more components to give a poorly soluble system whichis subject to no proven stoichiometry. In this case, a superposition ofinteractions occurs, mainly secondary valence bonds playing a part.

In the literature, poorly soluble peptide complexes are occasionallyalso described as a "salt". This description is likewise in many casesnot exact, since they are not, as already mentioned, substances having adefined composition.

In peptides and proteins ionic interactions admittedly occur, but theyare not responsible on their own for a structural or physical statechange.

For peptides and proteins, the term "complex" and "salt" is to be takenin a wider sense on account of the large number of functional groups,since several interactions which lead to synthesis and structure of thepeptides and proteins are superimposed.

Polyamine [sic] acids were used which are suitable as biophilic carriermaterials for peptides. It is essential to the invention here that theactive compounds are not bonded chemically to the polymer, but are onlyattached to the polymer by secondary valence bonds and hydrophobicinteractions.

Unexpectedly, it is seen that the LHRH antagonist cetrorelix especiallyhas a very high binding affinity to polyamino acids, in particular topolyglutamic acid and polyaspartic acid. Such a high affinity ofcetrorelix was not foreseeable on the basis of the literature up to nowand was surprising on the basis of the structure of the peptide.

The spontaneously precipitating complexes have a defined, reproduciblehormone content.

Should the hormone content in the complexes vary, however, and bedefined exactly, lyophilization has turned out as a suitable method.

These preparation conditions are significantly milder than described inearlier patents and thus prevent possible inactivation of the hormone.

The interactions occurring between the molecules on mixing the solutionslead to stable complexes which have a controllable active compoundrelease profile and an increased stability to proteolysis.

Polyamino acids thus affect not only the release-delaying behaviour, butsimultaneously offer protection from undesired, premature proteolyticdegradation. This aspect is especially of importance in view of thelong-term use of such preparations.

The release-delaying behaviour of the complexes can be significantlyaffected by the nature and the molecular mass of the polyamino acids,the incorporation of amino acids having hydrophobic side chains into thepolymer structure and by partial esterification of carboxyl groupspresent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows release curves in the random liberation system as afunction of molecular mass.

FIG. 2 shows release curves of cetrorelix complexes with methylpolyglutamate.

FIG. 3 shows release curves of Cetrorelix complexes with polyglutamicacid with leucine and phenylalanine.

FIG. 4 shows testosterone suppression in male rats after a single s.c.injection of 1. 5 mg/kg.

The invention is illustrated in greater detail with the aid of thefollowing working examples without, however, restricting it.

Preparation of polyamino acid-peptide complexes by precipitation

EXAMPLE 1

50 mg of polyamino acid are dissolved in 5 ml of H₂ O, in the case ofpoly-Glu with addition of 1 N NH₄ OH, gentle warming to 40° C. andultrasonic treatment. 50 mg of cetrorelix (as the acetate) are dissolvedin 4 ml of H₂ O. The polyamino acid solution is stirred and thecetrorelix solution is added in one step and then stored at 4° C. for 4h. Afterwards, the precipitate is centrifuged off at 4000 rpm for 5 min,the supernatant is removed and the precipitate is dried over P₂ O₅ invacuo for 24 hours. Since no stoichiometric complexes are present, theyield was based on the sum of the starting substances.

FIG. 1 shows various release curves in the random liberation system as afunction of the molecular mass (release medium: 0.01 m ammonium acetate,pH 7.0)

Yield: 50-65% of theory

Cetrorelix content in the complex: see Table 1

                  TABLE 1                                                         ______________________________________                                        Composition of the precipitated Cetrorelix-polyamino acid complexes                         Average  Hormone  Molar  Molar ratio                                                                                     molecular                                                   content in the    ratio  Hormone:                                                  mass   complex [%]   Hormone:                                               free carbox-                          Polyamino acid  [g/mol]    rel. error: 5%    PAS         yl groups          ______________________________________                                        Polyglutamic                                                                            5000     86         1:0.05 1:1.9                                      acid                                                                                               16000          85        1:0.016          1:2.1                                                                   50000                                                   60           1:0.02           1:8.2                                            Methyl poly-                              glutarnate                                                                    Degree of                                                                     methylation:                                                                  2.2%                 16000          81           1:0.02                                                          1:2.8                                      24.4%                16000          58           1:0.06                                                          1:6.7                                      Polyaspartic         7300           86           1:0.03                                                          1:2.2                                      acid                                                                                               14000          78           1:0.03                                                          1:3.8                                                           28000          69        1:0.023           1:6.1                                             Poly[(Glu,Phe)/    45000                                                     65        1:0.017           1:4.5                                              4:1]                                      Poly[(Glu,Leu)/    70000          79        1:0.005           1:2.4                                               4:1]                                    ______________________________________                                    

Preparation of polyamino acid-peptide complexes having a defined peptidecontent by lyophilization

EXAMPLE 2

Cetrorelix complex having a 50% peptide content 50 mg of polyamino acidare dissolved in 5 ml of H₂ O, in the case of poly-Glu with addition of1 N NH₄ OH, gentle warming to 40° C. and ultrasonic treatment. 50 mg ofcetrorelix (as the acetate) are dissolved in 4 ml of H₂ O. The polyaminoacid solution is stirred and the cetrorelix solution is added all atonce and stirred for a further 2 min. The resulting complex is frozen at-20° C. and subsequently lyophilized. Since no stoichiometric complexesare present, the yield was based on the sum of the starting substances.

Yield: 90-95% of theory

Cetrorelix content in the complex: 45-50%

EXAMPLE 3

By appropriate modification of the amount of polyamino acid andcetrorelix, a 70% cetrorelix complex was prepared analogously

EXAMPLE 4

An increase in the hydrophobicity, connected with an increase in therelease-delaying behaviour, can be achieved, inter alia, by the partialesterification of carboxyl groups. In FIG. 2, the release curves ofcetrorelix complexes with methyl polyglutamates are shown. FIG. 3 showsthe release curves of the Cetrorelix complexes of polyglutamic acid withleucine and phenylalanine.

EXAMPLE 5

For checking of the in vitro release experiments, cetrorelix-polyaminoacid complexes were tested in an animal experiment.

What is concerned here is the cetrorelix complexes with the followingpolyamino acids:

Polyglutamic acid, M: 5000 g/mol

Polyglutamic acid, M: 16000 g/mol

Polyaspartic acid, M: 7300 g/mol

In FIG. 4, testosterone suppression in male rats is shown after a singles.c. injection of 1.5 mg/kg. 5 animals were tested per experimentalgroup.

Using these results, it was possible to show that the complexesinvestigated have a long-term effect in testosterone suppression over600 hours. (FIG. 4)

The activity and suitability of the cetrorelix-polyamino acid complexesin principle as a depot preparation was confirmed.

What is claimed is:
 1. A process for the preparation of immobilized,activity stabilized, parenterally administerable peptide hormonepreparations from complexes of LHRH antagonists with polyamino acidscomprising the step of precipitating the peptide hormone complex fromaqueous solution.
 2. The process of claim 1, wherein the polyamino acidis polyglutamic acid or polyaspartic acid of average molecular masses2000-20000 g/mol.
 3. The process of claim 1, wherein the polyaminoacid-peptide hormone complex is further lyophilized.
 4. The processaccording to claim 1, wherein the release rate of the active compound iscontrolled by incorporation of hydrophobic amino acids into the polymerstructure or by partial esterification.